Antibacterial Effects of Ramulus mori Oligosaccharides against Streptococcus mutans

Abstract

Ramulus mori has been widely used in traditional Chinese medicine because of its physiological activities, including antibacterial, anti-inflammatory, and antioxidant activities. Antimicrobial properties of Ramulus mori extract have been well described. However, no information is available regarding on Ramulus mori oligosaccharides (RMOS). The aim of this study was to investigate the effects of RMOS on the growth and virulence properties of the cariogenic bacterium Streptococcus mutans. The effects of RMOS on the biofilm structure and virulence gene expression of S. mutans were also evaluated, and the results were compared with the effects of commercial prebiotic galactooligosaccharides. RMOS were found to have an antibacterial effect against S. mutans, resulting in significant reductions in acid production, lactate dehydrogenase activity, adhesion, insoluble extracellular polysaccharide production, glucosyltransferase activity, and biofilm formation in a dose-dependent manner. Moreover, the biofilm structure was visibly damaged. A quantitative real-time PCR assay revealed downregulation of virulence gene-regulated acid production, polysaccharide production, adhesion, biofilm formation, and quorum sensing. These findings suggest that RMOS may be a promising natural product for the prevention of dental caries.

Keywords: Ramulus mori oligosaccharides; Streptococcus mutans; anti-caries; antibacterial; biofilm.

Figure 1

Growth curve of S. mutans with different concentrations of RMOS. RMOS at 1/8 MIC (3.00 mg/mL); 1/4 MIC (6.00 mg/mL); 1/2 MIC (12.00 mg/mL), and the MIC (24.00 mg/mL) for 24 h incubation. Bacteria incubated without RMOS served as a negative control, and a culture medium with the MIC (24.00 mg/mL) of GOS served as a positive control. OD = optical density.

Figure 2

Effects of RMOS on the acidogenicity of S. mutans. (A) Glycolytic pH drop of S. mutans. (B) Relative activity of LDH of S. mutans. S. mutans was inoculated with RMOS at 1/8 MIC (3.00 mg/mL); 1/4 MIC (6.00 mg/mL); 1/2 MIC (12.00 mg/mL), and the MIC (24.00 mg/mL) and cultured anaerobically for 24 h. Bacteria incubated without RMOS served as a negative control, and a culture medium with the MIC (24.00 mg/mL) of GOS served as a positive control. Data are presented as means ± SD over three repetitions. Bars with various letters differ significantly (p < 0.05).

Figure 3

Effects of RMOS on the synthesis of IEPS of S. mutans. (A) IEPS production by S. mutans. (B) Relative activity of GTF of S. mutans. S. mutans was inoculated with RMOS at 1/8 MIC (3.00 mg/mL); 1/4 MIC (6.00 mg/mL); 1/2 MIC (12.00 mg/mL), and the MIC (24.00 mg/mL) and anaerobically cultured for 24 h. Bacteria incubated without RMOS served as a negative control, and culture medium with the MIC (24.00 mg/mL) of GOS served as a positive control. Data are presented as means ± SD over three repetitions. Bars with various letters differ significantly (p < 0.05).

Figure 4

Effects of RMOS on the biofilm of S. mutans. (A) Adhesion of S. mutans. (B) Formation of S. mutans biofilm. S. mutans was inoculated with RMOS at 1/8 MIC (3.00 mg/mL), 1/4 MIC (6.00 mg/mL), 1/2 MIC (12.00 mg/mL), and the MIC (24.00 mg/mL) and cultured anaerobically for 24 h. Bacteria incubated without RMOS served as a negative control, and a culture medium with the MIC (24.00 mg/mL) of GOS served as a positive control. Data are expressed as means ± SD of three repeats. Bars with different letters differ significantly (p < 0.05).

Figure 5

Effects of RMOS on biofilm structures of S. mutans. (A) SEM images of S. mutans. (B) CLSM images of S. mutans. (C) The percentage of viable bacteria in the biofilm. S. mutans was inoculated with RMOS at the MIC (24.00 mg/mL) and anaerobically cultured for 24 h. Bacteria incubated without RMOS served as a negative control, and a culture medium with the MIC (24.00 mg/mL) of GOS served as a positive control. The microstructure of the S. mutans biofilm on glass coverslips was observed at 1000×, 5000×, and 10,000× magnification by SEM. Biofilms were stained by STYO9 and PI and observed by CLSM. Live S. mutans was marked in green, and dead S. mutans was marked in red. Three separate experiments’ representative data are shown in the photographs. Data are expressed as means ± SD of three repeats. Bars with different letters differ significantly (p < 0.05).

Figure 6

Effects of RMOS on the expression of virulence genes of S. mutans. S. mutans was inoculated with RMOS at the MIC (24.00 mg/mL) and anaerobically cultured for 24 h. Bacteria incubated without RMOS served as a negative control, and culture medium with the MIC (24.00 mg/mL) of GOS served as a positive control. Data are expressed as means ± SD of three repeats. Statistical significance is identified as * p < 0.05, ** p < 0.01.